The present invention relates to composite scaffolds capable of supporting growth of complex tissue and to methods of manufacturing and using same.
Traditional medical treatments for functional deficiencies in organs have focused on using pharmaceutical compositions for replacing such functional deficiencies. However, in some cases, pharmaceutical replacement therapy cannot be instated since organ function is oftentimes complex and/or not completely understood.
In such cases, the only viable alternative is surgical replacement of the non-functional organ, however, in most cases, organ transplantation requires continuous use of immunosuppressive agents to prevent immunological rejection of the organ, depriving the patient of the full protective function of the immune system.
Moreover, the need for donor organs far exceeds the supply. Organ shortage has resulted in new surgical techniques, such as splitting adult organs for transplant. Despite fairly good results, such techniques still suffer from a lack of donor tissue.
The lack of viable donor tissue has led to the emergence of methods directed at generating engineered tissue for use in replacement procedures. Such methods typically employ a physical matrix for generating a three dimensional ex-vivo cell culture (see, for example, U.S. Pat. Nos. 4,060,081; 4,485,097; 4,458,678; 4,520,821; 5,041,138; 5,786,217; 5,855,610; and 6,143,293).
The basic concept of tissue engineering employs a scaffold (matrix) which provides a support upon which seeded cells can organize and develop into desired tissue prior to implantation. The scaffold provides an initial biomechanical profile for the replacement tissue until the cells can produce an adequate extracellular matrix. During the formation, deposition and organization of the newly generated matrix, the scaffold is either degraded or metabolized, eventually leaving engrafted tissue in its place.
Typically scaffolds are manufactured from biocompatible materials such that implantation thereof does not result in an adverse immune response or induced toxicity. In addition, scaffolds are typically manufactured with predetermined porosity so as to facilitate loading thereof of drugs or nutrients useful in promoting the growth of implanted cells.
Scaffold Manufacturing Approaches:
Fabrication of an appropriate scaffold, is determined by the type of tissue to be generated. Various scaffold manufacturing procedures rely on fabrication and casting of polymeric foams. Most of the polymeric foams used for tissue engineering applications are made from polylactides (PLA), polyglycolides) (PGA), or a combination of the two (PLGA).
Development of polymers, especially biodegradable polymers that produce non-toxic degradation products, as well as processing techniques to prepare porous three dimensional scaffolds with highly interconnected pore networks has become an important area of research.
Fiber bonding: Fiber bonding is a technique commonly for preparing structural interconnecting fiber networks for organ implants. Utilizing this process non-woven fibers are bonded together by immersing a non-bonded fiber structure of polymer A, such as PGA with a solution of polymer B (e.g., poly-L-lactic acid) (PLLA) using a solvent which does not dissolve polymer A. The solvent is then allowed to evaporate. The composite consisting of polymer A fibers embedded in a matrix of polymer B is heated above the melting temperature of polymer A to bond the fibers at their cross-points, and then polymer B is selectively dissolved (Mikos, et al. 1993 J. Biomed. Matl. Res. 27:183-189). The resultant bonded fiber structure of Polymer A has substantial rigidity, but the number of pores and their distribution is limited by that of the fiber mesh used in the fabrication.
Solvent-casting and particulate-leaching: In this technique, sieved salt particles, such as sodium chloride crystals, are spread in a PLLA/chloroform solution which is then used to cast a membrane. After evaporating the solvent, the PLLA/salt composite membranes are heated above the PLLA melting temperature and then quenched or annealed by cooling at controlled rates to yield amorphous or semi-crystalline forms with regulated crystallinity. The salt particles are eventually leached out by selective dissolution to produce a porous polymer matrix (Mikos, et al. 1992 Biodegradable Materials Research Society Symposium Proceedings, 252:352-358). However, the maximum level of porosity in this process is limited due to the difficulty of suspending salt particulates in the polymer solution. Furthermore, the crystalline structure of the sodium chloride salts gives rise to sharp edges which line the pores of the resulting foam, substantially reducing cell growth within the pores. Some of these problems can be overcome by making thin films of the foam and laminating them. Nevertheless, complex shaped implants cannot be easily compacted and the process is rather time-consuming.
Melt molding: Melt molding uses a Teflon™ mold, a mixture of fine PLGA powder and gelatin microspheres is heated above the glass-transition temperature of the polymer. The PLGA-gelatin composite is then removed from the mold and gelatin microspheres are leached out by selective dissolution in distilled de-ionized water.
Many of the above-described scaffold fabrication techniques generate scaffolds with inherent limitations.
Since such techniques require the use of severe heat or chemical treatment steps, cell seeding cannot be initiated during scaffold fabrication. In addition, chemical treated scaffolds can often invoke an inflammatory response following implantation.
Vascularization of an Engineered Tissue:
The need for a vascular network in engineered tissue has been demonstrated in studies of hepatocytes transplantation (Mooney et al. 1997). which demonstrated that survival of hepatocytes transplanted in vivo can be as low as 10% several days following transplantation, for a lack of ample vascularization. Similar findings were observed with transplants of smooth muscle cells (Cohn et al. 1997, Colton 1995).
To date most methods aimed at increasing implant permeability to nutrients, growth factors and oxygen rely on passive diffusion or alternatively external lining of the implant with artificial blood vessels.
Although numerous scaffold designs suitable for generating engineered tissues are known in the art, tissues engineered using such scaffolds typically lack the full functional capabilities of natural tissues. This is due to the fact that such scaffolds are either incapable of generating complex tissues having a fully functional architecture (e.g., vascularized), or are incapable of supporting growth of complex tissues altogether.
There is thus a widely recognized need for, and it would be highly advantageous to have, scaffolds which can be used to generate complex tissue grafts, such as, for example, vascularized tissue grafts, either ex-vivo or in-vivo while being devoid of the limitations inherent to prior art scaffolds.